The focus of this research grant has been the study of cellular interactions that guide T lymphocyte development in the thymus. In our last competitive renewal application (1997), we had argued that to understand positive selection, a natural, i.e. physiological, ligand of positive selection had to be identified. We had hypothesized that this goal could be best achieved, if by design the number of peptide candidates was severely limited. We had therefore proposed to produce a T cell antigen receptor transgenic (TCRtrans+) mouse using a CD8 + cytotoxic T lymphocyte (CTL) cell line that recognized peptides presented on the non-classical MHC class Ib molecule H2-M3 wt. This experimental design was based on the rational that H2-M3 wt MHC molecules preferentially bound peptides carrying a formylated Methionine (fM) in the N-terminal position. We had predicted further that in the thymus an H2-M3 with restricted CTL was selected on endogenous fM-peptides, whose only source were the thirteen genes of the mitochondrial genome. As originally proposed, we established an H2-M3 wt-restricted CTL line (C10.4), identified its cognate fM peptide (AttM), produced a C10.4 TCR transgenic mouse (C10+) and defined its physiological ligand of positive selection as the ND1 fM-peptide (ND1). Having identified the positively selecting and the cognate ligand during the previous funding period, we now present experiments aiming further to analyze how recognition of these ligands is used to guide thymic selection. To be able to control thymocyte recognition events, we have now reduced the complexity of C10+ thymic selection model to a two-cell in vitro thymic selection system, in which cloned lines of thymic epithelial cells (TECs) supported the selection and maturation of double positive (DP) into single positive (SP) thymocytes. We will provide evidence that this C10+ in vitro thymic selection model offers unique opportunities to define how thymic recognition events are translated into the development of T cells. More specifically, we are proposing to investigate how antigen recognition, accessory cell functions, signal delivery and thymocyte maturation states are integrated to guide the selection, maturation and lineage commitment of alpha/beta-T lymphocytes in the thymus. Since it has not been established that thymic selection of MHC class II-restricted T lymphocytes fully mimicked that of MHC class I-restricted T lymphocytes, we will adapt our in vitro thymic selection model to be used with H-2Ek-restricted AND TCRtrans+ (AND+) T cells.